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PubMed1993

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana.

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79-80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.

Amino Acid Sequence↗
PubMed1983

Relationship of plasma tartrate resistant acid phosphatase to the bone isoenzyme of serum alkaline phosphatase in hyperparathyroidism.

In 46 patients with primary hyperparathyroidism, in 21 non-dialysed patients with advanced renal failure, and in 52 patients on hemodialysis, a significant positive correlation was found between bone isoenzyme of serum alkaline phosphatase and plasma tartrate resistant acid phosphatase. In primary hyperparathyroidism, a significant positive correlation was found between the radiological degree of osteodystrophy and the biochemical parameters of bone remodelling. After removal of the parathyroid adenoma, only the tartrate-resistant acid phosphatase decreased to normal limits. Plasma tartrate resistant acid phosphatase was most significantly influenced by serum immunoreactive parathyroid hormone levels. In chronic renal failure, bone isoenzyme of serum alkaline phosphatase was most significantly influenced by serum immunoreactive parathyroid hormone levels, by hypocalcemia and by duration of hemodialysis. The results confirm that in hyperparathyroidism the extent of the whole-body rates of bone resorption and formation are approximately equal. The biochemical parameters can be used for serial assessment of the course of the disease but are not specific for diagnosis.

Acid Phosphatase↗
PubMed1987

Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A.

A 1.5 kb clone containing the full-length coding sequence of a type-1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type-1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1-101 of the type-1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.

Amino Acid Sequence↗
PubMed2006

Expression, purification and refolding of the phosphatase domain of protein phosphatase 1 (Ppt1) from Saccharomyces cerevisiae.

Here we report the recombinant expression of the catalytically active phosphatase domain of the Saccharomyces cerevisiae protein phosphatase 1 (Ppt1) in E. coli. Ppt1 consists of two domains: a 20 kDa TPR (tetratricopeptide repeat) domain, which mediates protein-protein interactions and directs Ppt1 to potential substrate proteins, e.g. the molecular chaperone Hsp90. The second, a 40 kDa phosphatase domain, exhibits catalytic activity and dephosphorylates phosphorylated serine/threonine residues of respective substrate proteins. The Ppt1 phosphatase domain was cloned and expressed in E. coli in unsoluble inclusion bodies. After isolating these, the aggregates were denatured with guanidinium hydrochloride and soluble protein was purified using affinity chromatography. Optimal renaturation conditions led to large amounts of the refolded phosphatase domain in high purity. Interestingly, further enzymatic studies revealed that the domain is not only correctly folded, but also shows higher catalytic activity compared to the full length protein.

Escherichia coli↗
PubMed2005

Structure of the hematopoietic tyrosine phosphatase (HePTP) catalytic domain: structure of a KIM phosphatase with phosphate bound at the active site.

Hematopoietic tyrosine phosphatase (HePTP) is a 38kDa class I non-receptor protein tyrosine phosphatase (PTP) that is strongly expressed in T cells. It is composed of a C-terminal classical PTP domain (residues 44-339) and a short N-terminal extension (residues 1-43) that functions to direct HePTP to its physiological substrates. Moreover, HePTP is a member of a recently identified family of PTPs that has a major role in regulating the activity and translocation of the MAP kinases Erk and p38. HePTP binds Erk and p38 via a short, highly conserved motif in its N terminus, termed the kinase interaction motif (KIM). Association of HePTP with Erk via the KIM results in an unusual, reciprocal interaction between the two proteins. First, Erk phosphorylates HePTP at residues Thr45 and Ser72. Second, HePTP dephosphorylates Erk at PTyr185. In order to gain further insight into the interaction of HePTP with Erk, we determined the structure of the PTP catalytic domain of HePTP, residues 44-339. The HePTP catalytic phosphatase domain displays the classical PTP1B fold and superimposes well with PTP-SL, the first KIM-containing phosphatase solved to high resolution. In contrast to the PTP-SL structure, however, HePTP crystallized with a well-ordered phosphate ion bound at the active site. This resulted in the closure of the catalytically important WPD loop, and thus, HePTP represents the first KIM-containing phosphatase solved in the closed conformation. Finally, using this structure of the HePTP catalytic domain, we show that both the phosphorylation of HePTP at Thr45 and Ser72 by Erk2 and the dephosphorylation of Erk2 at Tyr185 by HePTP require significant conformational changes in both proteins.

Amino Acid Motifs↗
PubMed1986

The human red cell acid phosphatase is a phosphotyrosine protein phosphatase which dephosphorylates the membrane protein band 3.

Human red cell cytosol acid phosphatase activity is supported by a main enzyme which can be extracted by DEAE and phosphocellulose chromatography. It uses pNPP as a substrate and is a protein phosphatase specific to phosphotyrosine. It dephosphorylates the tyrosine-phosphorylated cytosolic fragment of membrane protein 3. When taken together, these results suggest that the physiological role of red cell acid phosphatase is the FB3 phosphotyrosine dephosphorylation. Whatever it may be phosphotyrosine protein phosphatase activity is the first role of red cell acid phosphatase to be demonstrated.

Acid Phosphatase↗
PubMed2003

Influence of immobilization stress on the expression and phosphatase activity of protein phosphatase 2A in the rat brain.

BACKGROUND: Protein phosphatase 2A (PP2A) is a major kinase phosphatase that plays an important role in regulating the activities of protein kinase cascades. It has been revealed that stress changes neuronal gene expression by activating these cascades. We examined the expression of the catalytic subunit C and serine and threonine phosphatase activity of PP2A in the rat frontal cortex and hippocampus following various immobilization stress paradigms. METHODS: Immunoblot and immunohistochemical analyses were performed to examine the expression of PP2A. The level of phosphatase activity of PP2A was determined as the amount of free phosphate generated from a synthetic phosphopeptide. RESULTS: Immunoblot analysis revealed no significant change in the level of PP2A immunoreactivity in response to either a single or repeated stress. Immunohistochemical analysis revealed that neither a single nor repeated stress changed PP2A immunoreactivity in the hippocampus; however, the levels of serine and threonine phosphatase activity in the frontal cortex and hippocampus were significantly upregulated in response to a single or repeated stress. CONCLUSIONS: These results demonstrated that both a single and repeated immobilization stress upregulated the activity of PP2A in the rat brain, suggesting that PP2A may be involved, at least in part, in the downregulation of protein kinase activation induced by stress.

Animals↗
PubMed2002

Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes.

In adipocytes, protein kinase B (PKB) has been suggested to be the enzyme that phosphorylates phosphodiesterase 3B (PDE3B), a key enzyme in insulin's antilipolytic signalling pathway. In order to screen for PKB phosphatases, adipocyte homogenates were fractionated using ion-exchange chromatography and analysed for PKB phosphatase activities. PKB phosphatase activity eluted as one main peak, which coeluted with serine/threonine phosphatases (PP)2A. In addition, adipocytes were incubated with inhibitors of PP. Incubation of adipocytes with 1 microM okadaic acid inhibited PP2A by 75% and PP1 activity by only 17%, while 1 microM tautomycin inhibited PP1 activity by 54% and PP2A by only 7%. Okadaic acid, but not tautomycin, induced the activation of both PKBalpha and PKBbeta. Finally, PP2A subunits were found in several subcellular compartments, including plasma membranes (PM) where the phosphorylation of PKB is thought to occur. In summary, our results suggest that PP2A is the principal phosphatase that dephosphorylates PKB in adipocytes.

Adipocytes↗
PubMed1996

Stimulation of phosphorylase phosphatase activity of protein phosphatase 2A1 by protamine is ionic strength dependent and involves interaction of protamine with both substrate and enzyme.

The effects of protamine on the phosphorylase phosphatase activity of porcine cardiac protein phosphatase 2A1 (PP2A1) were complex and ionic strength dependent. Under ionic strength conditions that protamine activation was optimal, activation of PP2A1 by either dilution or heparin was prevented. A time-dependent deactivation of the protamine-stimulated phosphatase activity was observed when PP2A1 was preincubated with protamine. Protamine forms a very tight association with phosphorylase a, which is optimal at a 1:1 protamine:phosphorylase a monomer molar ratio. Protamine activation of PP2A1 activity, however, is not substrate-directed since the basic polypeptide did not stimulate either the activity of the catalytic subunit or trypsinolysis of [32P]phosphorylase a. The interaction of protamine with phosphorylase a does not apparently involve the phosphorylation site in the protein substrate (ser 14). The activation of PP2A1 by protamine is proposed to involve part of the basic polypeptide, not associated with phosphorylase a monomer, interacting with the regulatory and/or the catalytic subunit(s) of the phosphatase. A minimal model for the activation of PP2A1 by protamine was tested kinetically. In this model, free PP2A1 binds with decreasing affinities to the protamine:phosphorylase a complex, free phosphorylase a, and free protamine. Protamine decreases the K(m) of PP2A1 for the phosphorylase a monomer 5-fold and increases the Vmax 17-fold. Interaction of free protamine with PP2A1 inhibits the phosphatase activity.

Animals↗
PubMed2004

An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator.

We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.

Animals↗
PubMed1996

Dephosphorylation of catalytic subunit of cAMP-dependent protein kinase at Thr-197 by a cellular protein phosphatase and by purified protein phosphatase-2A.

Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.

Animals↗
PubMed1997

Purification of a fatty acid-stimulated protein-serine/threonine phosphatase from bovine brain and its identification as a homolog of protein phosphatase 5.

An arachidonic acid-stimulated Ser/Thr phosphatase activity was detected in soluble extracts prepared from rat pituitary clonal GH4C1 cells, rat or bovine brain, and bovine heart. The enzyme activity was purified to homogeneity from bovine brain as a monomer with a Mr of 63,000 and a specific activity of 32 nmol of Pi released per min/mg of protein when assayed in the presence of 10 microM phosphocasein in the absence of lipid. Arachidonic acid stimulated activity 4-14-fold, with half-maximal stimulation at 50-100 microM, when assayed in the presence of a variety of phosphosubstrates including casein, reduced carboxamidomethylated and maleylated lysozyme, myelin basic protein, and histone. Oleic acid, linoleic acid, and palmitoleic acid also stimulated activity; however, saturated fatty acids and alcohol or methyl ester derivatives of fatty acids did not significantly affect activity. The lipid-stimulated phosphatase was identified as the bovine equivalent of protein phosphatase 5 or a closely related homolog by sequence analysis of proteolytic fragments generated from the purified enzyme. When recombinant rat protein phosphatase 5 was expressed as a cleavable glutathione S-transferase fusion protein, the affinity-purified thrombin-cleaved enzyme exhibited a specific activity and sensitivity to arachidonic acid similar to those of the purified bovine brain enzyme. These results suggest that protein phosphatase 5 may be regulated in vivo by a lipid second messenger or another endogenous activator.

Amino Acid Sequence↗
PubMed1998

Association of the myosin-binding subunit of myosin phosphatase and moesin: dual regulation of moesin phosphorylation by Rho-associated kinase and myosin phosphatase.

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. We found that in MDCK epithelial cells, MBS accumulated at the tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling area, where moesin, a member of the ERM (ezrin, radixin, and moesin) family, was localized. Neither membrane ruffling nor an accumulation of moesin and MBS at the free-end plasma membrane was induced when MDCK cells were stimulated with TPA after the microinjection of C3, which ADP-ribosylates and inactivates Rho. MBS was colocalized with moesin at the cell-cell contact sites in MDCK cells. We also found that moesin was coimmunoprecipitated with MBS from MDCK cells. Recombinant MBS interacted with the amino-terminal domains of moesin and ezrin. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward moesin, which was phosphorylated by Rho-kinase. The phosphatase activity was inhibited when MBS was phosphorylated by Rho-kinase. These results suggest that MBS is recruited with moesin to the plasma membrane and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of moesin downstream of Rho.

ADP Ribose Transferases↗
PubMed2004

Essential role for the myotubularin-related phosphatase Ymr1p and the synaptojanin-like phosphatases Sjl2p and Sjl3p in regulation of phosphatidylinositol 3-phosphate in yeast.

The requirement of Vps34p, the sole phosphatidylinositol (PI) 3-kinase in Saccharomyces cerevisiae, for protein sorting to the vacuole in yeast has exemplified the essential role for phosphoinositides, phosphorylated derivatives of PI, in membrane trafficking. To better understand mechanisms that regulate PI 3-phosphate [PI(3)P]-mediated signaling, the role of the yeast myotubularin-related PI(3)P phosphatase Ymr1p was investigated. We found that Ymr1p and the synaptojanin-like phosphatase Sjl3p function as key regulators of the localization and levels of PI(3)P. Our data indicated that the ymr1Delta sjl3Delta double mutant aberrantly accumulated PI(3)P and demonstrated a steady-state redistribution of this lipid that leads to enrichment on the vacuolar membrane. This resulted in vacuole protein sorting defects, vacuolar fragmentation, and the misregulation of PI(3)P-specific effectors. Triple deletion of YMR1, SJL2, and SJL3 was lethal, suggesting an essential requirement for phosphatase-mediated PI(3)P regulation. Consistent with this, growth was restored to a ymr1Delta sjl2Delta sjl3Delta triple mutant by a PI(3)P-targeted Sac1p domain chimera (GFP-Sac1DeltaC-FYVE(EEA1)) that returned PI(3)P to levels comparable with wild-type cells. Together, this study demonstrated that Ymr1p, a myotubularin phosphatase family member, functions in the control of PI(3)P-dependent signaling and the maintenance of endosomal system integrity. In addition, this work defined an essential overlapping role for lipid phosphatases in the regulation of 3' phosphoinositides in yeast.

Aminopeptidases↗
PubMed1994

Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases.

A multiple sequence alignment of 44 serine/threonine-specific protein phosphatases has been performed. This reveals the position of a common conserved catalytic core, the location of invariant residues, insertions and deletions. The multiple alignment has been used to guide and improve a consensus secondary-structure prediction for the common catalytic core. The location of insertions and deletions has aided in defining the positions of surface loops and turns. The prediction suggests that the core protein phosphatase structure comprises two domains: the first has a single, beta sheet flanked by alpha helices, while the second is predominantly alpha helical. Knowledge of the core secondary structures provides a guide for the design of site-directed-mutagenesis experiments that will not disrupt the native phosphatase fold. A sequence similarity between eukaryotic serine/threonine protein phosphatases and the Escherichia coli diadenosine tetraphosphatase has been identified. This extends over the N-terminal 100 residues of bacteriophage phosphatases and E. coli diadenosine tetraphosphatase. Residues which are invariant amongst these classes are likely to be important in catalysis and protein folding. These include Arg92, Asn138, Asp59, Asp88, Gly58, Gly62, Gly87, Gly93, Gly137, His61, His139 and Val90 and fall into three clusters with the consensus sequences GD(IVTL)HG, GD(LYF)V(DA)RG and GNH, where brackets surround alternative amino acids. The first two consensus sequences are predicted to fall in the beta-alpha and beta-beta loops of a beta-alpha-beta-beta secondary-structure motif. This places the predicted phosphate-binding site at the N-terminus of the alpha helix, where phosphate binding may be stabilised by the alpha-helix dipole.

Acid Anhydride Hydrolases↗
PubMed1997

Protein phosphatase beta, a putative type-2A protein phosphatase from the human malaria parasite Plasmodium falciparum.

Protein phosphatases play a critical role in the regulation of the eukaryotic cell cycle and signal transduction. A putative protein serine/threonine phosphatase gene has been isolated from the human malaria parasite Plasmodium falciparum. The gene has an unusual intron that contains four repeats of 32 nucleotides and displays a high degree of size polymorphism among different strains of P. falciparum. The open reading frame reconstituted by removal of the intron encodes a protein of 466 amino acids with a predicted molecular mass of approximately 53.7 kDa. The encoded protein, termed protein phosphatase beta (PP-beta), is composed of two distinct domains. The C-terminal domain comprises 315 amino acids and exhibits a striking similarity to the catalytic subunits of the type-2A protein phosphatases. Database searches revealed that the catalytic domain has the highest similarity to Schizosaccharomyces pombe Ppa1 (58% identity and 73% similarity). However, it contains a hydrophilic insert consisting of five amino acids. The N-terminal domain comprises 151 amino acid residues and exhibits several striking features, including high levels of charged amino acids and asparagine, and multiple consensus phosphorylation sites for a number of protein kinases. An overall structural comparison of PP-beta with other members of the protein phosphatase 2A group revealed that PP-beta is more closely related to Saccharomyces cerevisiae PPH22. Southern blots of genomic DNA digests and chromosomal separations showed that PP-beta is a single-copy gene and is located on chromosome 9. A 2800-nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage (gametocytes). The results indicate that PP-beta may be involved in sexual stage development.

Amino Acid Sequence↗
PubMed1992

Alkaline phosphatase and phosphotyrosine phosphatase activities of cultured amniotic cells with trisomy 18.

In cultured amniotic cells from fetuses with Edward's syndrome (trisomy 18), the activities of two protein phosphatases, alkaline phosphatase and phosphotyrosine phosphatase, were measured. Comparison with normal fetal cells showed a different behavior for each enzyme. Alkaline phosphatase was significantly lowered while phosphotyrosine phosphatase remained at normal levels. The interest of these enzyme assays in the screening procedure of this severe chromosome defect is discussed.

Alkaline Phosphatase↗
PubMed1992

The pho-2A mutant of Neurospora crassa which is deficient in Pi-repressible alkaline phosphatase (EC 3.1.3.1) is also defective in Pi-repressible acid phosphatase (EC 3.1.3.2).

1. The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively. 2. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t1/2 = 4 min at 54 degrees C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54 degrees C, pH 5.4). 3. The pho-2A mutant of N. crassa secreted a more thermolabile acid phosphatase (t1/2 = 30 min at 50 degrees C, pH 5.4) than the wild strain (t1/2 of at least 80 min at 50 degrees C, pH 5.4). 4. The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t1/2 = 37 min at 54 degrees C, pH 5.4) than the wild strain in high Pi medium (t1/2 = 14 min at 54 degrees C, pH 5.4). 5. The pleiotropic nature of the pho-2 locus and its possible involvement in the mechanism of phosphatase secretion by N. crassa are proposed.

4-Nitrophenylphosphatase↗